Phoenixine-20-amide does not bind heterologously expressed GPR173

GPR173 (SREB3) is an orphan GPCR of the ‘Superconserved Receptors Expressed in the Brain’ family with high mRNA levels expressed in the hypothalamus, cortex and cerebellum. Phoenixin-20-amide (P20A), an endogenous hypothalamic ligand regulating ovarian cyclicity (Ullah et al., 2017; Stein et al., 2016), has been suggested to activate GPR173. In vitro studies on gonadotrophs showed that P20A stimulates GPR173, thereby increasing cAMP production, resulting in an elevated hypothalamic ‘Gonadotrophin Releasing Hormone Receptor’ (GnRHR) and kisspeptine expression, eventually increasing luteinizing hormone (LH) secretion (Treen et al., 2016; Stein et al., 2016). However, using the Promega GloSensor™ Technology, we failed to observe GPR173 mediated changes of cyclic AMP concentrations in HEK293 cellular models upon stimulation with P20A. Furthermore, it has been suggested that GPR173 could be stimulated by GnRH1_5 to trigger the recruitment of arrestin-3 in neurons co-expressing GnRHR, thereby inhibiting developmental neuronal migration (Larco et al., 2013). In our hands, with an arrestin-3-based firefly luciferase complementation assay in heterologously tranfected HEK293 cells, GPR173 was not able to recruit arrestin-3 upon stimulation with neither P20A nor GnRH1_5. Finally, GPR173 did neither bind any promiscuous g-proteins upon stimulation with P20A nor show any constitutive activity. Whether P20A influences the secretion of LH has not been studied, but P20A did not directly activate GPR173 in none of our assays. Nevertheless, transfected cellular models should be distinguished from native systems. Further studies are therefore necessary to determine if an endogenous partner is missing in our models and might be responsible for the previously reported results

Somatostatin Analogue Resistance in McCune-Albright Syndrome

McCune-Albright Syndrome (MAS) is a disorder characterized by involvement of multiple tissues, including skin, bone and endocrine glands. MAS is caused by post-zygotic mosaicism for GNAS gene mutations, which lead to a constitutively active form of the Gαs protein and increased cAMP levels. In the somatotropes of the pituitary, this increased cAMP leads to excessive growth hormone (GH) secretion. Somatostatin analogues (SSA), such as octreotide, that binds to somatostatin receptor SSTR2 are used in MAS patients to reduce GH secretion. However, MAS patients generally do not respond well to SSA, and GH control can rarely be achieved with SSA alone. In an in vitro model of MAS, five of the most common activating GNAS mutations lead to an increased level of cAMP, and a resistance to the treatment with SSA due to hyperactivated phosphokinase A. To further determine the underlying molecular causes of the SSA resistance, we studied the phosphorylation status of Filamin A (FLNA), a protein that couples somatostatin receptors to their cytoplasmic partners and to the cytoskeleton. Preliminary results suggest that three mutations of the GNAS gene may increase the phosphorylation status of FLNA

SOMATOSTATIN ANALOGUE RESISTANCE IN X-LAG SYNDROME - IDENTIFICATION OF THE UNDERLYING MECHANISMS

INTRODUCTION | X-Linked Acro-Gigantism (X-LAG) Syndrome is a rare genetic disorder caused by a microduplication on the X-chromosome of an array of genes comprising gpr101. This disorder leads to a pituitary adenoma secreting high levels of growth hormone, via elevated cAMP levels inside the somatotrophs of the adenoma. However, X-LAG patients cannot be treated by somatostatin analogues (SSA), such as octreotide, and present only a minor decrease in cAMP in response to pasireotide. METHODS & RESULTS | In heterologously transfected GH3 cells, cAMP levels do not decrease in presence of GPR101 in response to SSA treatment because the coupling of Gαi-proteins to the somatostatin receptors expressed in the pituitary adenoma (i.e., SSTR2 and SSTR5) is decreased in presence of GPR101. Moreover, due to a decreased arrestin-coupling to SSTR2 in presence of GPR101, SSTR2 is less endocytosed after stimulation. CONCLUSION | GPR101 quenches the signalling pathways of SSTR2 and SSTR5. However, further studies are necessary to determine the underlying cause of the patients’ resistance to SS